Name: drosophila passive avoidance behavior as a new paradigm to study associative aversive learning
Uploaded: 2021-10-15T15:00:00
Description: Watch this Scientific Journal Video about Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning at JoVE.com

This study was supported in parts by NIH R15ES029673 (AKM).

1. Preparation of passive avoidance apparatus
<ol>
	<li>Drill a 4 mm hole perpendicular to the wall surface of the 14 mL polypropylene culture tube and 8 mm away from the tube bottom. 
	NOTE: Use an electric drill and 5/32 drill bit for best results.</li>
	<li>Using a steel utility knife, cut off the upper part of the 14 mL polypropylene culture tube to create a 45 mm long tube bottom fragment. The bottom fragment serves as the lower compartment.</li>
	<li>Cut off the tip of 1,000 &#181;L blue pip...

<ol>
	<li>Kandel, E. R., Dudai, Y., Mayford, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+systems+biology+of+memory.">The molecular and systems biology of memory.</a> Cell. 157 (1), 163-186 (2014).</li><li>McGuire, S. E., Deshazer, M., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty+years+of+olfactory+learning+and+memory+research+in+Drosophila+melanogaster.">Thirty years of olfactory learning and memory research in Drosophila melanogaster.</a> Progress in Neurobiology. 76 (5), 328-347 (2005).</li><li>Livingstone, M. S., Sziber, P. P., Quinn, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+calcium/calmodulin+responsiveness+in+adenylate+cyclase+of+rutabaga,+a+Drosophila+learning+mutant.">Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant.</a> Cell. 37 (1), 205-215 (1984).</li><li>Quinn, W. G., Sziber, P. P., Booker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+memory+mutant+amnesiac.">The Drosophila memory mutant amnesiac.</a> Nature. 277 (5693), 212-214 (1979).</li><li>Dudai, Y., Jan, Y. N., Byers, D., Quinn, W. G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dunce,+a+mutant+of+Drosophila+deficient+in+learning.">dunce, a mutant of Drosophila deficient in learning.</a> Proceedings of the National Academy of Sciences of the United States of America. 73 (5), 1684-1688 (1976).</li><li>Busto, G. U., Cervantes-Sandoval, I., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+learning+in+Drosophila.">Olfactory learning in Drosophila.</a> Physiology. 25 (6), 338-346 (2010).</li><li>Tully, T., Quinn, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+and+retention+in+normal+and+mutant+Drosophila+melanogaster.">Classical conditioning and retention in normal and mutant Drosophila melanogaster.</a> Journal of Comparative Physiology. A: Sensory, Neural, and Behavioral Physiology. 157 (2), 263-277 (1985).</li><li>Wright, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+techniques+used+in+studying+associative+olfactory+learning+and+memory+in+adult+Drosophila+in+vivo:+A+historical+and+technical+perspective.">Evolution of the techniques used in studying associative olfactory learning and memory in adult Drosophila in vivo: A historical and technical perspective.</a> Invertebrate Neuroscience. 14 (1), 1-11 (2014).</li><li>Vogt, K., Yarali, A., Tanimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+stimulus+timing+in+visual+conditioning+leads+to+memories+with+opposite+valence+in+Drosophila.">Reversing stimulus timing in visual conditioning leads to memories with opposite valence in Drosophila.</a> PloS One. 10 (10), 0139797 (2015).</li><li>Koemans, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+courtship+conditioning+as+a+measure+of+learning+and+memory.">Drosophila courtship conditioning as a measure of learning and memory.</a> Journal of Visualized Experiments. (124), e55808 (2017).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> Journal of Visualized Experiments. (49), e2504 (2011).</li><li>Bozler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systems+level+approach+to+temporal+expression+dynamics+in+Drosophila+reveals+clusters+of+long+term+memory+genes.">A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes.</a> Plos Genetics. 13 (10), 1007054 (2017).</li><li>Atucha, E., Roozendaal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+avoidance+discrimination+task+to+investigate+accuracy+of+memory.">The inhibitory avoidance discrimination task to investigate accuracy of memory.</a> Frontiers in Behavioral Neuroscience. 9, 60 (2015).</li><li>Thiels, E., Hoffman, E. K., Gorin, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+behavioral+assay+for+the+assessment+of+sustained+photophobia+in+mice.">A reliable behavioral assay for the assessment of sustained photophobia in mice.</a> Current Eye Research. 33 (5), 483-491 (2008).</li><li>Detrait, E. R., Hanon, E., Dardenne, B., Lamberty, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+avoidance+test+optimized+for+discovery+of+cognitive+enhancers.">The inhibitory avoidance test optimized for discovery of cognitive enhancers.</a> Behavior Research Methods. 41 (3), 805-811 (2009).</li><li>Piper, M. D. W., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+ageing.">Drosophila as a model for ageing.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (9), 2707-2717 (2018).</li><li>Chalmers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicomponent+screen+for+feeding+behaviour+and+nutritional+status+in+Drosophila+to+interrogate+mammalian+appetite-related+genes.">A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes.</a> Molecular Metabolism. 43, 101127 (2021).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental Gerontology. 40 (5), 386-395 (2005).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+tools+to+handle+fruit+flies-Drosophila+melanogaster.">Simple homemade tools to handle fruit flies-Drosophila melanogaster.</a> Journal of Visualized Experiments. (149), e59613 (2019).</li><li>Barradale, F., Sinha, K., Lebestky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Drosophila+grooming+behavior.">Quantification of Drosophila grooming behavior.</a> Journal of Visualized Experiments. (125), e55231 (2017).</li><li>Denmark, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+chronic+social+defeat+stress+on+mouse+self-grooming+behavior+and+its+patterning.">The effects of chronic social defeat stress on mouse self-grooming behavior and its patterning.</a> Behavioural Brain Research. 208 (2), 553-559 (2010).</li><li>Kalueff, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+rodent+self-grooming+and+its+value+for+translational+neuroscience.">Neurobiology of rodent self-grooming and its value for translational neuroscience.</a> Nature Reviews: Neuroscience. 17 (1), 45-59 (2016).</li><li>Motulsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intuitive biostatistics: A nonmathematical guide to statistical thinking. Fourth edition. , (2018).</li><li>Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M., Syed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+analysis+of+long-term+grooming+behavior+in+Drosophila+using+a+k-nearest+neighbors+classifier.">Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier.</a> Elife. 7, 34497 (2018).</li><li>Mu, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+limbic+circuitry+involved+in+emotional+stress-induced+grooming.">A limbic circuitry involved in emotional stress-induced grooming.</a> Nature Communications. 11 (1), 2261 (2020).</li><li>Song, C., Berridge, K. C., Kalueff, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing'+rodent+self-grooming+for+neuroscience+research.">Stressing' rodent self-grooming for neuroscience research.</a> Nature Reviews: Neuroscience. 17 (9), 591 (2016).</li><li>Wang, C., Chan, J. S., Ren, L., Yan, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+reduces+cognitive+and+motor+functions+across+the+lifespan.">Obesity reduces cognitive and motor functions across the lifespan.</a> Neural Plasticity. 2016, 2473081 (2016).</li><li>Lewis, A. R., Singh, S., Youssef, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cafeteria-diet+induced+obesity+results+in+impaired+cognitive+functioning+in+a+rodent+model.">Cafeteria-diet induced obesity results in impaired cognitive functioning in a rodent model.</a> Heliyon. 5 (3), 01412 (2019).</li><li>Yohn, S. E., Galbraith, J., Calipari, E. S., Conn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+behavioral+and+neurocircuitry+disruptions+in+drug+addiction,+obesity,+and+binge+eating+disorder:+Focus+on+Group+I+mGluRs+in+the+mesolimbic+dopamine+pathway.">Shared behavioral and neurocircuitry disruptions in drug addiction, obesity, and binge eating disorder: Focus on Group I mGluRs in the mesolimbic dopamine pathway.</a> ACS Chemical Neuroscience. 10 (5), 2125-2143 (2019).</li><li>Lopez-Taboada, I., Gonzalez-Pardo, H., Conejo, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Diet:+Implications+for+brain+function+and+behavior.">Western Diet: Implications for brain function and behavior.</a> Frontiers in Psychololgy. 11, 564413 (2020).</li><li>Murashov, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+and+detrimental+effects+of+high+fat,+sugar,+and+salt+diet+in+wild-caught+Drosophila+simulans+are+reversed+by+flight+exercise.">Preference and detrimental effects of high fat, sugar, and salt diet in wild-caught Drosophila simulans are reversed by flight exercise.</a> FASEB Bioadvances. 3 (1), 49-64 (2021).</li><li>Krypotos, A. M., Effting, M., Kindt, M., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+learning:+A+review+of+theoretical+models+and+recent+developments.">Avoidance learning: A review of theoretical models and recent developments.</a> Frontiers in Behavioral Neuroscience. 9, 189 (2015).</li><li>Binder, M. D., Hirokawa, N., Windhorst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Neuroscience. , 3093 (2009).</li><li>Mery, F., Belay, A. T., So, A. K., Sokolowski, M. B., Kawecki, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+polymorphism+affecting+learning+and+memory+in+Drosophila.">Natural polymorphism affecting learning and memory in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (32), 13051-13055 (2007).</li><li>Tan, Y., Yu, D., Pletting, J., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gilgamesh+is+required+for+rutabaga-independent+olfactory+learning+in+Drosophila.">Gilgamesh is required for rutabaga-independent olfactory learning in Drosophila.</a> Neuron. 67 (5), 810-820 (2010).</li><li>&#214;gren, S. O., Stiedl, O., Stolerman, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Psychopharmacology. , 960-967 (2010).</li></ol>

Avoidance of threatening stimuli is a crucial characteristic of adaptive behavior in various species from C. elegance to human32. Avoidance learning procedures which typically entail the escaping of an aversive event, are commonly used behavioral tasks to investigate learning and memory processes in laboratory rodents13 since the 1970&#39;s32. In active avoidance procedures, an indifferent stimulus or conditioned signal (CS) is followed by a...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.
In the Representative Results, the legend for Figure 5 was updated from:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable increase in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
to:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable decrease in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
In the Discussion, the third paragraph&#160;was updated from:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly higher in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
to:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly lower&#160;in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.

Erratum: Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

Learning and memory is an evolutionarily ancient adaptation mechanism to the environment, conserved from Drosophila (D.) to human1. The fruit fly is a robust model organism to study fundamental principles of learning and memory as it offers a wide range of powerful genetic tools to dissect intrinsic molecular mechanisms2. The pioneering genetic screening studies, which identified rutabaga3, amnesiac4, and dunce5 genes critical for learning and memory2, took advantage of olfactory conditioning as the fruit flies rely on their keen sense of smell to find food, potential mates, and to avoid predators6.
Olfactory conditioning has become a popular paradigm to study the mechanism of learning and memory, thanks to the introduction of olfactory T-maze by Tully and Quinn7,8. Subsequently, other methods to measure various types of learning and memory have been proposed, including visual conditioning9, courtship conditioning10, aversive phototaxis suppression assay11, and wasp-exposure conditioning12. However, most of these assays have a complex setup that must be custom-built at a university workshop or purchased through a vendor. The paradigm described here is based on a simple behavioral assay to study aversive associative learning in flies that can be easily assembled with a few available supplies.
The described paradigm is equivalent to passive (or inhibitory) avoidance behavior in laboratory mice and rats in which animals learn to avoid a compartment where they have previously received electric foot shock13. In murids, the procedure is based on their innate avoidance of bright light and preference for darker areas14. On the first trial, the animal is placed into the bright compartment, from where the animal quickly exits, stepping into a dark compartment, where an electric foot shock is delivered. Usually, a single trial is sufficient to form a solid long-term memory, resulting in significantly increased latency 24 h later. The latency is then used as an index of the ability of the animal to remember the association between the aversive stimulus and the specific environment15.
This work describes an analogous procedure using D. as a model system which offers several advantages over rodent models including cost-effectiveness, larger sample size, the absence of regulatory oversight, and access to powerful genetic tools16,17. The procedure is based on negative geotaxis behavior, which manifests in flies&#39; urge to climb up when they are placed on a vertical surface18. The setup consists of two vertical chambers. On the first trial, a fruit fly is placed into a lower compartment. From there, it usually exits within 3-15 s, stepping into the upper compartment where it receives an electric shock. During a 1 min trial, some flies may occasionally re-enter the upper compartment, which results in an additional electric shock. During the testing phase, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first day indicating that flies formed aversive associative memory about the upper compartment. The latency, number of shocks, and the duration and frequency of grooming bouts are then used to analyze the animal behavior and the ability to form and remember the association between the aversive stimulus and the specific environment. The representative results reveal that exposure to the Western diet (WD) significantly impairs passive avoidance behavior in male flies, suggesting that the WD profoundly impacts the fly&#39;s behavior and cognition. Conversely, flight exercise alleviated the negative effect of the WD, improving passive avoidance behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bloomington Formulation diet</td>
			<td>Nutri-Fly</td>
			<td>&#160;66-112</td>
			<td>Available from Genesee Scientific Inc., San Diego, CA</td>
		</tr>
		<tr>
			<td>1000 &#181;L Blue tip</td>
			<td>Fisher</td>
			<td>NC9546243</td>
			<td></td>
		</tr>
		<tr>
			<td>17 x 100 mm 14 mL polypropylene culture tube</td>
			<td>VWR</td>
			<td>&#160;60818-689</td>
			<td></td>
		</tr>
		<tr>
			<td>Aduino-based Automatic Kontrol Module</td>
			<td>In-house</td>
			<td>AKM-007</td>
			<td>This unit is optional. Complete description, schematics, wiring diagram and a code are provided at the ECU Digital Market - https://digitalmarket.ecu.edu/akmmodule</td>
		</tr>
		<tr>
			<td>Dual-Display 2-Channel&#160; Digital Clock/Timer</td>
			<td>Digi-Sense</td>
			<td>AO-94440-10</td>
			<td>https://www.amazon.com/Cole-Parmer-AO-94440-10-Dual-Display-2-Channel-Jumbo-Digit/dp/B00PR0809G/ref=sr_1_5?dchild=1&#38;keywords=Dual-Display+timer+jumbo&#38;qid=1627660660&#38;sr= 
			8-5#customerReviews</td>
		</tr>
		<tr>
			<td>Electronic Finger Counter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://www.amazon.com/gp/product/B01M8IRK6F/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;psc=1</td>
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			<td>Fisherbrand Sparkleen 1 Detergent</td>
			<td>Fisher Scientific</td>
			<td>04-320-4</td>
			<td></td>
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		<tr>
			<td>Fly mouth aspirator</td>
			<td>In-house</td>
			<td></td>
			<td>Prepared as described in reference 19.</td>
		</tr>
		<tr>
			<td>Grass S88 stimulator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Could be replaced with any stimulator which can provide described parameters</td>
		</tr>
		<tr>
			<td>Kim-wipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>Kimberly-Clark Professional 34120</td>
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		<tr>
			<td>Metal block for fly immobilization</td>
			<td>In-house</td>
			<td></td>
			<td>4 x 13 x 23.5cm aluminum block</td>
		</tr>
		<tr>
			<td>Nutiva USDA Certified Organic, non-GMO, Red Palm Oil</td>
			<td>Nutiva</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Nutiva-Certified-Cold-Filtered-Unrefined-Ecuadorian/dp/B00JJ1E83G/ref=sxts_rp_s1_0?cv_ct_cx=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&#38;dchild=1&#38;keywords=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&#38;pd_rd_i=B00JJ1E83G&#38;pd_ 
			rd_r=f35e9d2f-afe4-44b6-afc2-1c9cd705be18&#38;pd_rd_w= 
			R3Zb4&#38;pd_rd_wg=eUv1m&#38;pf_rd_ 
			p=c6bde456-f877-4246-800f-44405f638777&#38;pf 
			_rd_r=M94N11RC7NH333EMJ66Y 
			&#38;psc=1&#38;qid=1627661533&#38;sr=1-1-f0029781-b79b-4b60-9cb0-eeda4dea34d6</td>
		</tr>
		<tr>
			<td>Shock tube</td>
			<td>CelExplorer</td>
			<td>TMA-201</td>
			<td>https://www.celexplorer.com/product_detail.asp?id=217&#38;MainType=110&#38;SubType=8</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Accusplit</td>
			<td>A601XLN</td>
			<td>https://www.amazon.com/gp/product/B0007ZGZYI/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;psc=1</td>
		</tr>
		<tr>
			<td>Transparent vinyl tubing (3/4&#8221; OD, 5/8&#8221; ID)</td>
			<td>Lowes</td>
			<td></td>
			<td>Avaiable from Lowes</td>
		</tr>
	</tbody>
</table>

drosophila passive avoidance behavior as a new paradigm to study associative aversive learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

The passive avoidance was studied in D. melanogaster (Canton-S) and D. simulans. The experiments compared the latencies and number of received shocks between consecutive trials. Initially, the experiments were performed with 3-4 day old male D. melanogaster flies. Flies were maintained on the standard Bloomington Formulation diet in a climate-controlled environment at 24 &#176;C under a 12 h light-dark cycle, 70% humidity, and controlled population density. The density was controlled b...

Watch this Scientific Journal Video about Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning at JoVE.com

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

This protocol describes a simple behavioral assay that allows for studying adverse associative learning and memory consolidation in flies, including effects of gene environment interactions. The assay presented here is a simple, reproducible, and cost effective procedure for studying memory mechanisms that can be easily assembled with a few available supplies. This assay could provide insight into basic mechanisms underlying learning and memory impairments resulting from various genetic, pharmacological, and dietary manipulations.Demonstrating this procedure will be Morgan Tedder and Steven Bradley, undergraduate students from my laboratory. To begin, drill a four millimeter hole perpendicularly about four millimeters from the bottom within a 14 milliliter polypropylene culture tube. Remove the upper part of the culture tube to make a 45 millimeter bottom fragment utilized as the lower compartment.Cut off the top of a 1000 microliter pipette tip to create a 12 millimeter fragment. Insert the fragment into the four millimeter hole of the culture tube as a loading dock to transfer the flies. Cut a 15 millimeter piece of transparent vinyl tubing and insert the lower and upper compartment from opposite ends into the tubing.Attach the assembly vertically using a two-prong adjustable clamp. Locate the upper compartment vertically attached with shock tubes. Connect the shock tubes with an electrical stimulator to generate electric shocks.Using an ice cold block, immobilize three-to-four-day-old flies and transfer them into separate vials with food 24 hours before the experiment. Assign a code to all the vials as described in the text manuscript. Gently suck one fly into the mouth aspirator by drawing air.Deposit the fly by lightly blowing into the loading dock and immediately start a one-minute timer and stopwatch. Turn on the stimulator and deliver an electric shock when the fly enters the shock tube. Press the stopwatch to record the first latency.If the fly reenters the shock tube, deliver additional shocks. Using a tally or an Arduino-based counter, record the number of shocks during a one-minute trial. Gently transfer the fly back into the vial at the end of a one-minute trial.Record the latency, the number of received shocks, and any notable change in behavior. Using 70%ethanol, clean the lower and shock compartment. Wipe with a lint-free cleaning tissue and dry it using a hair dryer.Repeat the trial with the next fly. Clean the lower compartment with water and odorless detergent after completing the experiment. Using 70%ethanol, wipe the lower and shock compartment and air dry overnight.By repeating the above process after 24 hours, perform a second trial. In a similar sequence, test the flies. After performing a passive avoidance assay in Drosophila melanogaster male flies, results have shown that the latency increases, the number of shocks decreases, demonstrating that flies learned association between the upper compartment and an electric shock.Frequency distribution revealed that most flies enter the upper compartment and receive one to three shocks in the first trial. By the fourth trial, most flies do not enter the upper compartment and receive zero shocks. In subsequent experiments, the trial duration was reduced to one minute because the frequency distribution for latencies clearly showed that if the fly does not enter the upper compartment within 60 seconds in the first trial, it usually does not enter at all.The passive avoidance assay experiments were repeated in Drosophila simulans male and female flies. Results have shown that the flies were effective at learning the passive avoidance behavior as evident from the changes in frequency distributions for latencies and shocks in male and female flies. Comparison of latencies and number of shocks between male and female did not reveal any statistically significant differences.However, female flies received slightly more shocks in each trial. Analysis of grooming bouts in trials two and three revealed that the total duration of grooming decreases significantly in female flies from trial one to trial three. This suggests that flies experience behavioral stress which parallels rodent anxiety-like behaviors.Comparison of the effects of the Western diet and flight exercise on passive avoidance behavior show that the Western diet decreases the latency and increases the number of shocks indicating that the Western diet impairs aversive associative learning in flies. Conversely, flight exercise mitigated the negative effect of the Western diet. When attempting this procedure, remember not to stress flies.Stressed flies would either enter the upper compartment too quickly or won't enter at all. All experiments should be performed under the same environmental conditions and at the same time of the day. This technique gives researchers a new simple assay that can be used to dissect intrinsic mechanisms of learning and memory consolidation in flies.

Research

JoVE Journal

Behavior

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A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions

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How to Study Placebo Responses in Motion Sickness with a Rotation Chair Paradigm in Healthy Participants

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Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

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Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

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Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

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A Methodology for Capturing Joint Visual Attention Using Mobile Eye-Trackers

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency ...

Experimental Paradigm for Measuring the Effect of Induced Emotion on Grammar Learning

Previous studies have found that emotion has significant influence on the learning of foreign language vocabulary and textual comprehension. However, ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...